Hydrogen exchange is a powerful method for studying structural and dynamic properties in biological macromolecules. It can be used to understand hydrogen-bonding, solvent accessibility and seconday structure elements in addition to other related features (01PROG135 ). Several approaches can be used:

• Exchange with Deuterium. The sample is lyophilized, redissolved in D₂O, and several ¹H-¹⁵N HSQC or similar experiments are recorded at several times after sample preparation. The following can occurs:
• Fast-exchange protons: are not observable upon the addition of D₂O. It is typical when secondary structure is not present.
• Medium-exchange protons: are observable during some minutes.
• Slow-exchange protons: are observable during some hour/days.
• Very slow-exchange protons: are observable after days.

Such proton-deuterium substitution experiments can determine exchange rates well below 0.1-0.01 s^-1.
• Temperature and pH dependence of NH proton chemical shifts (Amide proton Temperature Coefficients)
• Qualitative amide-proton exchange rates can be measured from the cross-section of amide proton-water exchange peak taken of NOESY-type experiments (such a 2D NOESY or EXSY, ¹⁵N-edited NOESY....), acquired without water presaturation, at the F₁ chemical shift of the water. Thus amide protons exchanging rapidly with solvent shown intense exchange cross-peaks.
• Measurement of diffusion coeficients. Thus amide protons with fast exchange are largely attenuated using mild conditions. Relative NH lifetimes can also be obtained from this approach ( 99JB25 ).
• Examples:
  Hydrogen-deuterium exchange and pH studies on the blue-shifted intermediate of photoactive yellow protein ( 00BIO14392 )